Background
Cellular compartmentalization is essential for the regulation of
metabolism and gene expression (Harrington, Feliu, Wiuf, & Stumpf,
2013). Reciprocal communication between the mitochondria, chloroplasts
and nuclei is not only vital for the efficient functions of these
compartments, but it also ensures the rapid adjustment of their protein
content and composition to changing environmental conditions.
Mitochondria-derived and plastid-derived retrograde signals are
therefore important components in the regulation of nuclear gene
expression (Diaz et al., 2018; Grubler et al., 2017; Kindgreen et al.,
2012; Pogson, Woo, Forster, & Small, 2008). Retrograde signalling
between the chloroplast and nucleus are not easily distinguished from
those that operate during the proplastid-to-chloroplast transition in
leaf development because of crossover between the biogenic and
operational control of chloroplast functions (Pogson et al., 2008). In
plastids, transcription is under the control of two types of RNA
polymerases, a unique eubacterial-type plastid-encoded polymerase (PEP)
and phage-type nucleus-encoded polymerases (NEPs). These RNA-polymerases
specifically regulate the transcription of different subsets of genes
but can also co-regulate a portion of the plastidial genes. The
formation of chloroplasts from proplastids requires the establishment of
the PEP complex. The PEP complex, which is composed of a catalytic core
comprised of plastid-encoded proteins (rpoA, rpoB, rpoC1 andrpoC2 ) and additional polymerase-associated proteins (PAP)
including other nuclear-encoded polymerase-associated proteins and sigma
factors (SIGs), which are required by PEP for promoter recognition( Dietz, & Pfannschmidt, 2011). PEP status/activity provides
positive retrograde signals from the chloroplasts that convey essential
information to the nucleus to promote PhANG expression.
The WHY family of proteins, which are specific to the plant kingdom
(Desveaux et al., 2004) have a putative KGKAAL DNA binding domain that
allows binding to ssDNA molecules of differing nucleotide sequence
(Grabowski, Miao, Mulisch, & Krupinska, 2008) which may allow them to
function as PAPs allowing the possibility of a functional interaction
between these proteins (Días et al., 2017).
Mitochondria to nucleus signalling, which involves two key transcription
factors: ANAC013 and ANAC017 , is also linked to plastid to
nucleus signalling (Shapiguzov et al., 2019). The ANAC013 andANAC017 transcription factors are released from the endoplasmic
reticulum upon perception of appropriate signals and translocated to the
nucleus, where they activate the expression of a specific set of genes
called mitochondrial dysfunction stimulon (MDS) genes that include the
alternative oxidases, SOT12 and ANAC013 (De Clercq et al.,
2013; Safrany et al., 2008). The enhanced expression of ANAC013provides positive feedback regulation of the signalling pathway. The
nuclear-localised RADICAL-INDUCED CELL DEATH1 (RCD1) protein suppresses
ANAC013 and ANAC017 functions (Shapiguzov et al., 2019). In addition,
SOT12 belongs to the group of MDs genes that overlap with the genes
induced by the SAL1, 3’-phosphoadenosine 5’-phosphate (PAP) chloroplast
retrograde signalling pathway (Van Aken, & Pogson, 2017).
All plants have two WHY genes (WHY1 and WHY2 ).WHY1 encodes a protein that is located in chloroplasts and nuclei
while WHY2 encodes a mitochondria-targeted protein (Desveaux,
Maréchal, & Brisson, 2005). WHY1 protein interacts with thylakoid
membrane proteins and with the chloroplast nucleoids (Krupinska et al.,
2014; Melonek et al., 2010). Unlike many other species, Arabidopsis has
a third WHY gene, AtWHY3 that is targeted to plastids (Krause et
al., 2005). However, the intracellular localization of the WHY proteins
appears to be flexible and determined by developmental and environmental
signals. For example, the WHY2 protein that is primarily associated with
mitochondrial nucleoids, was found in mitochondria, chloroplasts and
nuclei during leaf senescence (Huang et al., 2020). Moreover, it appears
that WHY3 can compensate for WHY2 in the Arabidopsis why
2-1 mutant because WHY3 can be targeted to both chloroplasts and
mitochondria (Golin et al., 2020). The expression of WHY2 in Arabidopsis
decreased the expression of genes encoded by the chondriome (Maréchal et
al., 2008). Similarly, the expression of the tomato SlWHY2 in transgenic
tobacco plants led to mitochondrial gene transcription and stabilization
of mitochondrial functions (Zhao et al., 2018).
Barley leaves deficient in the WHY1 protein have higher levels of
chlorophyll than the wild type with an enhanced abundance of
plastome-encoded transcripts (Comadira et al., 2015; Krupinska et
al. , 2019). In contrast, the leaves of the Arabidopsis why1mutant and why1why3 double mutants are phenotypically similar to
the wild type. However, a why1why3polIb-1 triple mutant defective
in WHY1, WHY3, and the DNA polymerase 1B (Pol1B) exhibited a severe
yellow-variegated phenotype (Lepage, Zampini, & Brisson, 2013).
WHY1, WHY3 and RECA1 are
associated with the chloroplast RNase H1 AtRNH1C protein and work
together to maintain chloroplast genome integrity (Wang et al .,
2021). Maize transposon insertion lines in WHY1 (Zmwhy1-1) have
equivalent amounts of chloroplast DNA (cpDNA) to the wild type but are
deficient in plastid ribosomes resulting in an albino phenotype
(Prikryl, Watkins, Friso, van Wijk, & Barkam, 2008).
We have previously characterized the phenotypes, and metabolite and
transcriptome profiles of three RNAi-knockdown barley lines (W1-1, W1-7
and W1-9) that have very low levels of HvWHY1 expression
(Comadira et al., 2015). The formation of plastid ribosomes and the
establishment of photosynthesis was delayed in the RNAi-knockdown barley
lines (Krupinska et al., 2019). Given the growing number of reports
showing that WHY1 functions as a transcription factor in the nucleus,
regulating the expression of genes involved in a wide range of processes
including phytohormone synthesis, development and defence, we posed the
hypothesis that WHY1 in the nuclei of developing leaves could also
control chloroplast development. We therefore investigated the
intracellular distribution of WHY1 between proplastids and nuclei in the
bases of developing wild type barley leaves.
We also characterized the
transcript and metabolite profiles of barley lines (W1-1 and W1-7)
lacking WHY1. We discuss the data indicating that the observed delay in
plastid development in barley lines lacking WHY1 results from functions
of the protein in the nuclei as well as the plastids.